The present invention relates to chemical analysis systems and, more particularly to an analytical instrument in which sample components are separated by differential electrokinetic migration through a narrowbore capillary. A major objective of the present invention is to provide for post-separation mixing of the sample with another fluid to aid in identification and quantification of the separated sample components. An illustrative example is the post-separation addition of a fluorogenic labelling reagent to separated protein components prior to fluorescence detection.
Chemical analyses of complex organic structures has made noteworthy advances in biotechnology possible. Biotechnology has provided techniques for manufacturing life-supporting medicines and other products which would otherwise be in short supply if natural sources had to be relied upon. In addition, entirely new medical products are in development which may arrest and cure heretofore untreatable diseases. Biotechnology promises new products for agriculture which will feed the world's expanding populations and which will enhance the ability of famine-prone countries to sustain themselves.
Chemical analysis of biological samples generally involves the separation of the samples into components for identification and quantification. Capillary zone electrophoresis (CZE) is one of a class of methods in which the different components are moved within a narrowbore capillary at respective and different rates so that the components are divided into distinct zones. The distinct zones can be investigated within the capillary or outside the capillary by allowing the components to emerge from the capillary for sequential detection.
In CZE, a sample is introduced at an input end of a longitudinally extending capillary and moved toward an output end. Electrodes of different potentials at either of the capillary generate the electrical forces which move the sample components toward the output end of the capillary. This movement includes two distinct components, one due to electro-osmotic flow and the other due to electrophoretic migration.
Electro-osmotic flow results from charge accumulation at the capillary surface due to preferential adsorption of anions from the electrolyte solution which fills the capillary bore. The negative charge of the anions attracts a thin layer of mobile positively charged electrolyte ions, which accumulate adjacent to the inner surface. The longitudinally extending electric field applied between the ends of the capillary by the electrodes attracts these positive ions so that they are moved toward the negative electrode at the output end of the capillary. These positive ions, hydrated by water, viscously drag other hydrated molecules not near this inner wall, even those with neutral or negative net charge. The result is a bulk flow of sample and the containing electrolyte solution toward the output end of the capillary. Thus, electro-osmotic flow provides a mechanism by which neutral and negatively charged, as well as positively charged, molecules can be moved toward a negative electrode. Typically, a CZE capillary has a bore diameter of less than 200 .mu.m and preferable less than 100 .mu.m, to ensure that the outer molecules interact sufficiently with more central molecules to effect an electro-osmotic flow which is fairly uniform across the capillary cross section.
Superimposed on this electro-osmotic flow is the well known motion of charged particles in response to an electrical field, commonly referred to as electrophoretic migration. The electrolyte solution acts as the medium which permits the electric field to extend through the capillary between the electrodes. Positively charged molecules migrate toward the negative electrode faster than the mean flow due to electro-osmotic flow. Negatively charged molecules are repelled by the negative electrode, but this repulsion is more than compensated by the electro-osmotic flow. Thus, negatively charged sample molecules also advance toward the negative electrode, albeit more slowly than the positively charged molecules. Neutral molecules move toward the negative electrode at an intermediate rate governed by the electro-osmotic flow.
After a sufficiently long migration through the separation capillary, the different sample components separate into bands or zones due to the differential movement rates as a function of species-specific charge. An appropriately selected and arranged detector can detect these zones seriatim as they pass. Components can be identified by the time of detection and can be quantified by the corresponding detection peak height and/or area. In some cases, the bands can be collected in separate containers for a distinct identification and/or quantification process.
There are several types of detectors used to detect proteins in capillary separation systems. Ultraviolet absorbance (UV) detectors are among the most common. Other electro-magnetic absorbance detectors could be used. In addition, chemi-luminescence, refractive index and conductivity detectors have been used. All these methods lack the sensitivity required to detect many peaks in CZE protein analysis. High sensitivity is required because the quantity of the total sample is limited, and the detector must be capable of detecting components that make up only of fraction of the total sample. Limitations on sample quantity stem from the requirement that the sample be dissolved in electrolyte and that the concentration of the sample be low enough to avoid perturbation of the electrical field which would lead to distortion of the separated component zones. The sample quantity is further limited by the capillary bore diameter and by the necessity of confining the sample initially to a relative short longitudinal extent. The initial sample extent governs the minimum zone breadth and thus the ability of the system to resolve similarly charged sample components.
The detector must be able to detect small quantities of the component in each sample zone. A UV detection system is faced with low concentrations and very short illumination path lengths and typically yields a poor signal-to-noise ratio. Other detection methods are similarly limited. Thus, while CZE is effective in separating protein components, there has been a limitation in finding a sufficiently sensitive detector for identifying and quantifying the separated components.
Fluorescence detection has been applied in conjunction with liquid chromatography (LC), a class of alternative component separation techniques. In liquid chromatography, a liquid "mobile" phase ushers components through a capillary at different rates related to the component's partitioning between the mobile phase and a stationary phase. Zones thus form as a function of partitioning ratios. The zones can be illuminated and the resulting fluorescence detected. Few proteins can be detected with sufficient sensitivity using their intrinsic fluorescence. However, labelling reagents can be used to enhance protein fluorescence. A major advantage of using fluorescence detection is that the increased sensitivity required by small sample quantities can be achieved by using very intense illumination. Thus, fluorescence detection used with labelling reagents promises to enhance the ability to identify and quantify sample components.
Unfortunately, liquid chromatography is not well suited for high resolution separation of proteins. While partitioning ratios differ among components, the molecules of any one component at any given time will be divided between the mobile phase and stationary phase, and thus move at different rates from each other. Despite averaging effects over the length of the capillary, sufficient zone broadening is induced by the partitioning to prevent high resolution separation of protein components. Since its only source of zone broadening is longitudinal diffusion, CZE represents an approximately ten-fold improvement in zone-breadth-limited resolution over liquid chromatography.
Fluorescence detection of proteins is not used in conjunction with CZE for a number of reasons. As in liquid chromatography, use of the fluorescence intrinsic to proteins in not generally applicable. Preseparation fluorescence labelling is incompatible with CZE for several reasons. For example, pre-separation labelling of protein components causes same-species molecules to have different charges. Thus, one component separates into multiple peaks, rendering detections virtually uninterpretable. Furthermore, sensitivity problems are aggravated because each peak represents only a fraction of a sample component.
Post-separation labelling involves the introduction of fluorogenic labelling reagent after separation and before detection. Post separation mixing is addressed by Van Vliet et al, "Post-Column Reaction Detection for Open-Tubular Liquid Chromatography Using Laser-Induced Fluorescence", Journal of Chromatography, Vol. 363, pp. 187-198, 1986. This article discloses the use of a Y-connector for introducing reagent into the effluent of a separation capillary. One problem with the Y-connector is the inevitable turbulence that occurs as the streams merge at an oblique angle. The turbulence stirs the sample stream, severely broadening the component zones. This broadening can be tolerable in a low resolution system, but not in a high-resolution CZE system.
Post-separation mixing is also addressed by Weber et al. in "Peroxyoxalate Chemilumininescence Detection with Capillary Liquid Chromatography" in Analytical Chemistry, Vol. 59, pp. 1452-1457, 1987. Weber et al. disclose the use of a Teflon tube to convey the separated sample components emerging from a liquid chromatography capillary, packed with silica particles to the interior of a mixing capillary. An annular gap between the Teflon tube and the mixing capillary is used to introduce chemi-luminescence reagent coaxially of the sample emerging from the narrower (0.2 mm) Teflon tube and into the (0.63 mm) mixing capillary. (Note that chemi-luminescence can not be employed in protein component detection.) Turbulence is minimized since the reagent flow is fast enough to define a sheathing flow confining the sample. However, a problem with the sheathing flow is that mixing occurs slowly. Sufficient mixing of the chemi-luminescence reagent with sample components thus requires a relatively long mixing interval and large mixing volume, during which zone broadening in the absence of impairs resolution significantly. While this zone broadening may be tolerable in the relatively low resolution liquid chromatography system disclosed, it would negate the advantages of a high resolution CZE system.
Thus, one obstacle to post-separation fluorescence labelling in high resolution systems is the attainment of rapid, yet low-turbulence and low volume, mixing of reagent and sample. However, CZE and other electrokinetic separation techniques face another obstacle to post-separation introduction of fluorescence labelling reagents, as well as other fluids. Fluid introduction generally requires apertures and other material inhomogeneities in capillary walls defining the sample path. In a CZE separation system, these inhomogeneities can cause field perturbations which interfere with electro-osmotic and other electro-kinetic effects. At a minimum these perturbations cause zone broadening, but can even partially or completely impair electro-kinetic movement of sample components.
In summary, CZE provides a separation technique which affords the resolution required for the analysis of complex proteins, but lacks a sufficiently sensitive compatible detection technique. Fluorescence detection provides a desirable level of sensitivity, but the required labelling has not been workable in the CZE context. What is needed is a system which combines the resolving power of CZE with the detection sensitivity of available with fluorescence-labelled proteins.